Serial intracerebral passage of dengue type 1 or type 2 virus in mice was shown previously to select for mouse neurovirulent mutants which also proved to be attenuated for humans. In a similar manner a neurovirulent mutant of DEN4 (strain H241) was selected by serial intracerebral passage in mice. DEN4 H241 neurovirulent (N) replicated less efficiently than its DEN4 H241 parent (P) in simian LLC-MK2 cells. An intratypic DEN4 chimera containing the C-PreM-E structural protein genes from DEN4 H241N also exhibited marked restriction of growth in LLC-MK2 cells. Analysis of virion proteins of DEN4 H241N or its derived C-PreM-E chimera grown in mosquito C6/36 cells indicated that very little M was produced. There is considerable evidence that immature PreM-containing flavivirus virions are less infectious than the mature M-containing virus. For this reason, we constructed a panel of chimeras that contained one or more of the variant amino acids of the mutant C-PreM-E sequence substituting for the corresponding sequence of the parental virus. These chimeras were analyzed to determine if any of these mutations was responsible for inefficient PreM cleavage. Mutants which contained two substitutions in PreM, i. e., Lys165-Glu and Val188-Ala, with or without the Thr81-Ile substitution in C, exhibited reduced PreM cleavage. Reduction of PreM cleavage was also observed for chimeras which contain multiple mutations in E, i.e., Thr434-Ile, Ser435-Pro, and Phe680-Leu, with the exception of the chimera containing Thr434-Ile and Ser435-Pro. Efficient PreM cleavage was required for virus to achieve high level of replication in simian LLC-MK2 cells. Similarly, the genetic basis for the reduced replicative efficiency of the mouse-passaged MD-1 vaccine strain in LLC-MK2 cells compared to its parental DEN1 Hawaii strain was investigated. A combination of three substitutions in E independently caused restriction of replication in simian cells. A single Val207-Ala substitution in PreM was also independently responsible for restriction of replication of MD-1 or its derived chimera in LLC-MK2 cells. This PreM mutation is located immediately downstream of the N-terminus of M, which is cleaved from PreM by the host cell furin enzyme. The Val207-Ala mutation did not affect PreM cleavage in mosquito cells, but this mutation might possibly restrict PreM cleavage in simian cells and thus, be responsible for growth restriction.